Effects Of Low Concentration Quinolone On Physiological Characteristics And Proteome Expression Of Streptococcus Thermophilus

In recent years, low concentration of antibiotics are commonly detected in many animal derived food materials. When these materials are used to produce fermentation food, the lactic acid bacteria (LAB) in food may be stressed by the antibiotic residues. Meanwhile, both endo-and exogenous LAB in human intestine could be stressed during the antibiotic treatment. Low concentration of antibiotics can stimulate LAB to produce a series of stress response and prevent harmful substances from entering cells. However, the physiological function and fermentation performance of LAB should be further studied. Therefore, the aim of this study was to determine the effects of low concentration of quinolone on the physiological property, fermentation performance and proteome expression of S. thermophilus grx02 by microbial physiological, metabolomic and proteomic technologies. The results may be helpful to comprehensively evaluate the prebiotic properties and safety of LAB.1. The effects of low concentration of quinolone on physiological characteristics of S. thermophilus grx02The minimal inhibitory concentrations (MIC) of ofloxacin (OFX) and ciprofloxacin (CPFX) against S. thermophilus grx02 were determined. Then, S. thermophilus grx02 were, inoculated into MRS fluid medium containing 0,1/50,1/10 and 1/2 MIC of OFX or CPFX. The growth curve, bacterial morphology of S. thermophilus grx02 were determined. The effects of OFX and CPFX on key enzyme activities involved in intracellular lactose metabolism and membrane permeability were also investigated. Results showed that the MIC of OFX and CPFX against S. thermophilus grx02 were 7.75 and 3.89μg/mL, respectively. This indicated that LAB was moderately susceptible to OFX and CPFX. The growth rate of S. thermophilus grx02 was significantly reduced at 1/2 MIC of OFX and CPFX (P<0.05). The number of small and micro colonies increased obviously at different MIC concentrations. In addition, the bacterial length of antibiotic treatment group was elliptical and significantly shortened as compared to that of control group. The capsule of antibiotic treatment group was obviously thinner and denser than that of control group. The cell membrane permeability and the activities of β-galactosidase and lactate dehydrogenase decreased significantly at 1/10 and 1/2 MIC of OFX or CPFX, respectively (P<0.01). At 1/50 MIC of CPFX, the β-galactosidase activity decreased, however, the lactate dehydrogenase activity increased. These results indicate that S. thermophilus grx02 may initiate stress response with changing relevant physiological properties under the stress of quinolone.2. The effects of low concentration of quinolone on fermentation performance of S. thermophilus grx02The effects of different sub-MIC quinolone on curd time, acid and glue producing capacity, and proteolytic activity of S. thermophilus grx02 were studied. Meanwile, the effects of quinolone on texture properties, rheological behavior, sensory properties, viable count and volatile flavor substances of S. thermophilus grx02 fermented milk was also investigated. Results showed that the syneresis of S. thermophilus grx02 fermented milk was significantly strengthened, however, the adhesion, tackiness and chewiness were decreased at 1/2 MIC of OFX and CPFX. At the same time, the proteolytic activity and the viable count of fermented milk decreased significantly during storage at 4℃. The curd time of S. thermophilus grx02 fermented milk increased obviously, the acid-producing speed, viscosity, storage stability and rheological properties reduced significantly at 1/10 MIC of OFX and CPFX (P<0.05). The gel stability and resistance capability to mechanical movement and temperature change of S. thermophilus grx02 fermented milk were decreased. The sensory quality of the milk was significantly influenced by OFX and CPFX when their residual concentration was higher than 1/10 MIC, such as a little whey separation, deteriorated thick feeling and significant lower texture score. When the concentration of OFX and CPFX residue reached to 1/50 MIC, the content of total volatile compounds, such as diacetyl and organic acid contents obviously changed. These results suggested that the residual concentration of OFX and CPFX in the raw milk for processing fermented dairy product should be lower than 1/50 MIC.3. The effects of OFX on proteomic expression of S. thermophilus grx02The cytosolic proteins of S. thermophilus grx02 grown at middle logarithmic phase in MRS with or without 1/2 MIC of OFX were separated by two dimensional gel electrophoresis. The differentially expressed proteins were identified by MALD-TOF-MASS. At last, the function toxinomy of differentially expressed proteins and analyzed. The two dimensional gel electrophoresis profiles of S. thermophilus grx02 showed clear background with well-resolution and repeatability. Forty seven proteins were expressed differently in two profiles, and 38 of these proteins could be identified. Among the differentially expressed proteins,5 proteins disappeared in samples treated by 1/2 MIC of CPFX containing CspA, general stress protein 39, ATP synthase F1, uracil phosphoribosyltransferase, and nitroreductase. Thirty three proteins were down-regulated. They mainly participate in carbohydrates and energy metabolism, protein and nucleotide synthesis, and cellular energy level regulation. These results indicated that protein expression of S. thermophilus grx02 and the activity of enzymes in metabolic pathways are remarkablely inhibited by sub-MIC of CPFX. Therefore, the growth and metabolic rate of S. thermophilus may be reduced. This could be one of stress mechanisms of S. thermophilus grx02 to resist antibiotics.